The phenomenon of programmed cell death, or "apoptosis," is involved in and important to the normal course of a wide variety of developmental processes, including immune and nervous system maturation. Apoptosis also plays a role in adult tissues having high cell turnover rates (Ellis, R. E., et al., Ann. Rev. Cell. Biol. 7: 663-698 (1991); Oppenheim, R. W., Ann. Rev. Neurosci. 14: 453-501 (1991); Cohen, J. J., et al., Ann. Rev. Immunol. 10: 267-293 (1992); Raff, M. C., Nature 356: 397-400 (1992)). In addition to its role in development, apoptosis has been implicated as an important cellular safeguard against tumorigenesis (Williams, G. T., Cell 65: 1097-1098 (1991); Lane, D. P., Nature 362: 786-787 (1993)). Under certain conditions, cells die by apoptosis in response to high-level or deregulated expression of oncogenes (Askew, D., et al., Oncogene 6: 1915-1922 (1991); Evan, G. I., et al., Cell 69: 119-128 (1992); Rao, L., et al., Proc. Natl. Acad. Sci. USA 89: 7742-7746 (1992); Smeyne, R. J., et al., Nature 363: 166-169 (1993); Tanaka, S., et al., Cell 77: 829-839 (1994); Wu, X., et al., Proc. Natl. Acad. Sci. USA 91: 3602-3606 (1994)). Suppression of the apoptotic program, by a variety of genetic lesions, may contribute to the development and progression of malignancies. This is well illustrated by the frequent mutation of the p53 tumor suppressor gene in human tumors (Levine, A. J., et al., Nature 351: 453-456 (1991)).
Other factors have been identified which appear to play roles in regulating apoptosis. One of these, the Insulin-Like Growth Factor-I Receptor (IGF-IR), is a member of the tyrosine kinase family of signal transducing molecules. The IGF-IR is activated by the ligands IGF-I, IGF-II and insulin at supra-physiological concentrations, and plays an important role in the development, growth, and survival of normal cells (LeRoith, D., et al., Endocrine Revs. 16: 143-163 (1995); Lowe, W. L., Jr. "Biological actions of the Insulin-like growth factor receptors," in LeRoith, D., Ed., Insulin-like Growth Factors: Molecular and Cellular Aspects, CRC Press, Boca Raton, Pub. (1991); Baserga, R., et al., Cell Prolif. 27: 63-71 (1994)). Over-expression of the IGF-IR leads to the transformation of fibroblasts and conversely, IGF-IR null fibroblasts are refractory to transformation by a number of oncogenes (Sell, C., et al., Mol. Cell Biol. 14: 3604-3612 (1994)).
There is considerable evidence for a role for the IGF-IR in the maintenance of tumor cells in vitro and in vivo. IGF-IR levels are elevated in tumors of lung (Kaiser, U., et al., J. Cancer Res. Clin Oncol. 119: 665-668 (1993); Moody, T. W. and Cuttitta, F., Life Sciences 52: 1161-1173 (1993)), breast (Pollak, M. N., et al., Cancer Lett. 38: 223-230 (1987); Foekens, J. A., et al., Cancer Res. 49: 7002-7009 (1989) Cullen, K. I., et al., Cancer Res. 49: 7002-7009 (1990)) and colon (Remaole-Bennet, M. M., et al., J. Clin. Endocrinol. Metab. 75: 609-616 (1992); Guo, Y. S., et al., Gastroenterol. 102: 1101-1108 (1992)). Increased levels of IGF-I and/or IGF-II expression have been associated with human tumors (McCauley, V. M., et al., Cancer Res. 50: 2511-2517 (1990); Bhatavdekar, J. M., et al., Neoplasma 41: 101-103 (1994)). Many of these tumor cell types respond to IGF-I with a proliferative signal in culture (Nakanishi, Y., et al., J. Clin. Invest. 82: 354-359 (1988); Freed, K. A. and Herrington, A. C., J. Mol. Endocrinol. 3: 509-514 (1989)), and autocrine or paracrine loops for proliferation in vivo have been postulated (LeRoith, D., et al., Endocrine Revs. 16: 143-163 (1995); Yee, D., et al., Mol. Endocrinol. 3: 509-514 (1989)).
IGF-I protects from apoptosis induced by cytokine withdrawal in IL-3-dependent hemopoietic cells (Rodriguez-Tarduchy, G., et al., J. Immunol. 149: 535-540 (1992)), and from serum withdrawal in Rat-1/mycER cells (Harrington, E., et al., EMBO J. 13: 3286-3295 (1994)). Of cytokines present in fetal bovine serum, including the mitogens EGF and PDGF, IGF-I proved to be the most potent in inhibition of myc-induced death in Rat-1 cells. The anti-apoptotic function of IGF-I was evident in the post-commitment stage of the cell cycle and also in cells blocked in cell cycle progression by etoposide or thymidine.
The demonstration that c-myc driven fibroblasts are dependent on IGF-I for their survival suggests that there is an important role for the IGF-IR in the maintenance of oncogene driven tumor cells by specifically inhibiting apoptosis, a role distinct from the better characterized proliferative effects. This would be similar to a role thought to be played by other anti-apoptotic genes such as bcl-2 in promoting tumor survival (McDonnell, T. J., et al., Cell 57: 79-88 (1989); Hockenberry, D. M., et al., Nature 348: 334-336 (1990)). The protective effects of IGF-I are dependent upon receptor levels rather than on availability of the ligand (Resnicoff, M., et al., Cancer Res. 55: 3739-3741 (1995a)). Support for an anti-apoptotic function of IGF-IR in the maintenance of tumor cells was also provided by a study using antisense oligonucleotides to the IGF-IR that identified a quantitative relationship between IGF-IR levels, the extent of apoptosis and the tumorigenic potential of a rat syngeneic tumor (Rescinoff, M., et al., Cancer Res. 55: 3739-3741 (1995b)).
Fibroblasts from IGF-IR null mice have been used to demonstrate a requirement for the IGF-IR in transformation, and also to map domains in the receptor essential for the proliferative and transformation function of the IGF-IR. Specifically, the C-terminal region of the IGF-IR is required for the transformation function. Receptors which are truncated at amino acid 1229 fail to transform fibroblasts derived from IGF-IR null mice, but retain full proliferative activity (Surmacz, E., et al., Exp. Cell Res. 218: 370-380 (1995)). Within the C-terminal region, the transforming activity has been further localized to a domain between amino acids 1245 and 1294; substitution of the single tyrosine 1251 with phenylalanine impairs transformation function (Miura, M., et al., J. Biol. Chem. 270: 22639-22644 (1995b)), substitution of the four serines (1280-1283) completely abolishes transformation Li et al., J.Biol. Chem., 217:12254-12260 (1996), and substitution of histidine 1293 and lysine 1294 reduces transformation activity Hongo et al., Oncogene, 12:1231-1238 (1996). All of the transformation-defective, truncated and point mutant receptors retain proliferative capacity. These studies indicate that two separate functions of the receptor, proliferation and transformation, are spatially distinct within the receptor and that transformation may need additional signals to those required for proliferation. Mutations at the ATP binding site in the kinase domain, at the tyrosine cluster in the kinase domain, or at tyrosine 950 (the major binding site for well defined substrates of the IGF-IR, IRS-1 and SHC) abolish both proliferation and transformation (Miura, M., et al., Cancer Res. 55: 663-667 (1995a); Li, S., et al., J. Biol. Chem. 269: 32558-32564 (1994); Gronberg, M., et al., J. Biol. Chem. 268: 23435-23440 (1993)).
As the preceeding discussion demonstrates, while recent studies have advanced the general understanding of the transformation and proliferative functions of the IGF-IR in vertebrate cells, the apparent anti-apoptotic function of the IGF-IR remains less well characterized. Elucidation of IGF-IR domains involved in the receptor's anti-apoptotic function would be of great value in the development of compositions which modulate the survival of certain cells, such as cancer cells. The ability to modulate the anti-apoptotic activity of the IGF-IR would also allow the development of compositions and strategies for treating cells affected by diseases, such as neurodegenerative diseases, and by acute hypoxic injury, such in stroke, in which activation of the IGF-IR's anti-apoptotic function would be beneficial. Conversely, inactiviation of the anti-apoptotic function of the IGF-IR in tumor cells would be a useful and specific treatment strategy. Accordingly, a need persists to identify the potential domain(s) in the IGF-IR responsible for its anti-apoptotic function.